This invention relates to polypeptide hormone analogues that exhibits enhanced pharmaceutical properties, such as increased increased thermodynamic stability, augmented resistance to thermal fibrillation above room temperature, decreased mitogenicity, and/or altered pharmacokinetic and pharmacodynamic properties, i.e., conferring a biphasic time course of action relative to (a) a fast-acting component similar to soluble formulations of the corresponding prandial or wild-type human hormone and (b) a prolonged component similar to microcrystalline NPH formulations of wild-type insulin or insulin analogues. More particularly, this invention relates to insulin analogues consisting of a single polypeptide chain that (i) contains a novel class of foreshortened connecting (C) domains between A and B domains with acidic residues at the first and second positions, (ii) contains an amino-acid substitution at position A8, and (iii) contains an acidic residue at position A14. Of length 6-11 residues, the C domains of this class consist of an N-terminal acidic element and a C-terminal segment basic element similar to that of wild-type proinsulin. The single-chain insulina analogues of the present invention may optionally contain standard or non-standard amino-acid substitutions at other sites in the A or B domains, such as positions B28 and B29 known in the art to confer rapid action.
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. Naturally occurring proteins—as encoded in the genomes of human beings, other mammals, vertebrate organisms, invertebrate organisms, or eukaryotic cells in general—often confer multiple biological activities. A benefit of non-standard proteins would be to achieve selective activity, such as decreased binding to homologous cellular receptors associated with an unintended and unfavorable side effect, such as promotion of the growth of cancer cells. Yet another example of a societal benefit would be augmented resistance to degradation at or above room temperature, facilitating transport, distribution, and use. An example of a therapeutic protein is provided by insulin. Wild-type human insulin and insulin molecules encoded in the genomes of other mammals bind to insulin receptors is multiple organs and diverse types of cells, irrespective of the receptor isoform generated by alternative modes of RNA splicing or by alternative patterns of post-translational glycosylation. Wild-type insulin also binds with lower affinity to the homologous Type 1 insulin-like growth factor receptor (IGF-1R).
An example of a further medical benefit would be optimization of the pharmacokinetic properties of a soluble formulation such that the time course of insulin action has two phases, a rapid phase and a delayed phase (FIG. 1). Such a combination of rapid and delayed phases is known in the art to be conferred by mixtures of a solution of zinc insulin analogue hexamers (as provided by, but not limited to, insulin lispro and insulin aspart) with a micro-crystalline suspension of the same analogue prepared in combination with zinc ions and protamines or protamine-related basic peptides; the latter component is designated in the art as Neutral Protamine Hagedorn (NPH) micro-crystalline suspensions. Pre-mixed insulin products known in the art contain varying ratios of these two components, such as 25% soluble phase and 75% micro-crystalline phase, 30% soluble phase and 70% micro-crystalline phase, or 50% of each phase. Such pre-mixed products are widely used by patients with diabetes mellitus in the developing world due to their ease of use with reduction in the number of subcutaneous injections per day relative to the separate administration of a prandial (rapid-acting) insulin formulation (or prandial insulin analogue formulation) and of a NPH micro-crystalline suspension of wild-type insulin or insulin analogue. The simplification of insulin regimens of insulin provided by pre-mixed biphasic insulin products has also proven of benefit to patients with diabetes mellitus in affluent societies (i) for whom treatment solely by prandial insulin analogue formulations leads to suboptimal glycemic control or excessive weight gain, (ii) for whom treatment solely by NPH insulin products or basal insulin analog formulations leads to suboptimal glycemic control due to upward excursions in the blood glucose concentration within three hours after a meal, or (iii) patients of the above two classes for whom addition of an oral agent (such as metformin) does not result in satisfactory glycemic control.
Existing biphasic insulin products require a complex and costly method of manufacture due to the post-fermentation and post-purification steps needed to grow NPH micro-crystals. Further, such products suffer from an intrinsic susceptibility of both the soluble and micro-crystalline components to physical and chemical degradation above room temperature. The biphasic pharmacokinetic properties of these pre-mixed products may change with storage of the vials above room temperature due to interchange of insulin molecules between the soluble and micro-crystalline phases. Finally, the use of micro-crystalline suspensions can be associated with uncertainties in dosing as the number of micro-crystals drawn into a syringe can vary from withdrawal to withdrawal even from the same vial.
In light of the above disadvantages of existing biphasic insulin products, the therapeutic and societal benefits of biphasic insulin formulations would be enhanced by the engineering of insulin analogues whose pharmacokinetic properties as a mono-component soluble solution confer a biphasic pattern of insulin action. Additional benefits would accrue if the novel soluble insulin analogue were simpler and less costly to manufacture (i.e., by avoiding the requirement for micro-crystallization) and/or if the novel soluble insulin analogue were more refractory than wild-type insulin to chemical or physical degradation at or above room temperature. Such resistance to degradation above room temperature would be expected to facilitate use in regions of the developing world where electricity and refrigeration are not consistently available. The challenge posed by such degradation is deepened by the pending pandemic of diabetes mellitus in Africa and Asia. Because fibrillation poses the major route of degradation above room temperature, the design of fibrillation-resistant formulations may enhance the safety and efficacy of insulin replacement therapy in such challenged regions. Still additional therapeutic and societal benefit would accrue if the soluble biphasic insulin analogue should exhibit reduced mitogenicity in assays developed to monitor insulin-stimulated proliferation of human cancer cell lines.
Administration of insulin has long been established as a treatment for diabetes mellitus. A major goal of conventional insulin replacement therapy in patients with diabetes mellitus is tight control of the blood glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions below the normal range are associated with immediate adrenergic or neuroglycopenic symptoms, which in severe episodes lead to convulsions, coma, and death. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinapathy, blindness, and renal failure. Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The hormone is stored in the pancreatic-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B chain (residue B30) to the N-terminal residue of the A chain. A variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide. Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19) is thought to be coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin assembles to form soluble Zn2+-coordinated hexamers shortly after export from ER to the Golgi apparatus. Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). Individual residues are indicated by the identity of the amino acid (typically using a standard three-letter code), the chain and sequence position (typically as a superscript). Pertinent to the present invention is the invention of novel foreshortened C domains of length 6-11 residues, containing an N-terminal acidic motif and a C-terminal basic motif, in place of the 36-residue wild-type C domain characteristic of human proinsulin and in combination with amino-acid substitutions at positions A8 and A14 of the A chain and optionally at positions B28 and B29 of the B chain.
Fibrillation, which is a serious concern in the manufacture, storage and use of insulin and insulin analogues for the treatment of diabetes mellitus, is enhanced with higher temperature, lower pH, agitation, or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, patients with diabetes mellitus optimally must keep insulin refrigerated prior to use. Insulin exhibits an increase in degradation rate of 10-fold or more for each 10° C. increment in temperature above 25° C.; accordingly, guidelines call for storage at temperatures <30° C. and preferably with refrigeration. The NPH micro-crystalline component of existing biphasic insulin product is susceptible to fibrillation above room temperature as well as a distinctive mode of chemical degradation due to proteolytic cleavage within the A chain; such cleavage inactivates the insulin or insulin analogue.
The above cleavage of the insulin A chain in NPH micro-crystals is representative of a process involving the breakage of chemical bonds. Such breakage can lead to loss or rearrangement of atoms within the insulin molecule or the formation of chemical bonds between different insulin molecules, leading to formation of polymers. Whereas cleavage of the A chain in NPH micro-crystals is thought to occur on the surface of the folded state, other changes in chemical bonds are mediated in the unfolded state of the protein or in partially unfolded forms of the protein, and so modifications of insulin that augment its thermodynamic stability also are likely to delay or prevent chemical degradation. It is therefore a desirable property of an insulin analogue that its free energy of denaturation (as typically measured by circular dichroism at a helix-sensitive wavelength as a function of the concentration of a chemical denaturant) should be equal to or greater than that of wild-type insulin or equal to or greater than that of a prandial (rapid-acting) insulin analogue in current clinical use.
Insulin is also susceptible to physical degradation. The present theory of protein fibrillation posits that the mechanism of fibrillation proceeds via a partially folded intermediate state, which in turn aggregates to form an amyloidogenic nucleus. In this theory, it is possible that amino-acid substitutions that stabilize the native state may or may not stabilize the partially folded intermediate state and may or may not increase (or decrease) the free-energy barrier between the native state and the intermediate state. Therefore, the current theory indicates that the tendency of a given amino-acid substitution in the two-chain insulin molecule to increase or decrease the risk of fibrillation is highly unpredictable. Models of the structure of the insulin molecule envisage near-complete unfolding of the three alpha-helices (as seen in the native state) with parallel arrangements of beta-sheets forming successive stacking of B-chains and successive stacking of A-chains; native disulfide pairing between chains and within the A-chain is retained. Such parallel cross-beta sheets require substantial separation between the N-terminus of the A-chain and C-terminus of the B-chain (>30 Å), termini ordinarily in close proximity in the native state of the insulin monomer (<10 Å). Marked resistance to fibrillation of single-chain insulin analogues with foreshortened C-domains is known in the art and thought to reflect a topological incompatibility between the splayed structure of parallel cross-beta sheets in an insulin protofilament and the structure of a single-chain insulin analogue with native disulfide pairing in which the foreshortened C-domain constrains the distance between the N-terminus of the A-chain and C-terminus of the B-chain to be unfavorable in a protofilament. A ribbon model of a single-chain insulin analogue is shown in FIG. 2; a space-filling model of the insulin moiety is shown in FIG. 3 to highlight the role of the engineered connecting domain (C domain; stick representation in FIG. 3).
The present invention was motivated by the medical and societal needs to engineer a biphasic single-chain insulin analogue in a soluble and monophasic formulation at neutral pH intended for twice-a-day injection, i.e., on a schedule similar to that of current pre-mixed regular-NPH biphasic insulin products. Our single-chain design is intended to combine (i) resistance to degradation with (ii) substantial in vivo hypoglycemic potency with (iii) reduced cross-binding to IGF-1R and (iv) intrinsic biphasic pharmacokinetics and pharmacodynamics in the absence of a component consisting of a micro-crystalline suspension. It would be desirable, therefore, to invent single-chain insulin analogue that, as a soluble protein solution at neutral pH, exhibits biphasic pharmacokinetic- and pharmacodynamics properties on subcutaneous injection such that both rapid-onset of action and a prolonged tail of action are achieved leading to an overall profile that resembles those of premixed products as exemplied by “Humalog® Mix75/25” or “NovaMix® 30.” Biphasic insulin analog formulations of the present invention will therefore provide simplified twice-a-day bolus-basal regimens that will be of clinical advantage in the developed and developing world. Single-chain biphasic insulin analogue formulations may also be initiated in insulin-naïve patients not well controlled on metformin, a first-line oral agent widely used in the treatment of T2DM. The mechanism of biphasic action of current regular-NPH premixed products, based on pharmacokinetic properties of the two components, is shown in schematic form in FIG. 4A. While not wishing to be constrained by theory, a possible mechanism of biphasic pharmacokinetics by analogies of the present invention is shown in schematic form in FIG. 4B.
We envisage that the products of the present invention will disproportionately benefits patients in Western societies whose compliance with more complex regimens is uncertain. It is known in the art that health-care outcomes—and long-term adherence to prescribed regimens in chronic diseases such as T2DM and the metabolic syndrome—are a complex function of socioeconomic status, formal education, family structure, and cultural belief systems. Indeed, these societal issues are of increasing concern given the growing burden of obesity and T2DM among under-represented minorities, including African-Americans, Hispanic and indigenous Americans. Single-chain biphasic insulin analogue formulations of the present invention are therefore intended to benefit insulin-requiring T1DM and T2DM patients who have inadequate glycemic control with basal-only insulin therapy but for whom a full basal-bolus regimen is impractical.